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. 2017 Dec 21:11:17-27.
doi: 10.2147/OTT.S147316. eCollection 2018.

Quercetin suppresses DNA double-strand break repair and enhances the radiosensitivity of human ovarian cancer cells via p53-dependent endoplasmic reticulum stress pathway

Cheng Gong  1 , Zongyuan Yang  1 , Lingyun Zhang  2 , Yuehua Wang  2 , Wei Gong  2 , Yi Liu  3
Affiliations

Quercetin suppresses DNA double-strand break repair and enhances the radiosensitivity of human ovarian cancer cells via p53-dependent endoplasmic reticulum stress pathway

Cheng Gong et al. Onco Targets Ther. .

Abstract

Quercetin is proven to have anticancer effects for many cancers. However, the role of tumor suppressor p53 on quercetin's radiosensitization and regulation of endoplasmic reticulum (ER) stress response in this process remains obscure. Here, quercetin exposure resulted in ER stress, prolonged DNA repair, and the expression of p53 protein; phosphorylation on serine 15 and 20 increased in combination with X-irradiation. Quercetin pretreatment could potentiate radiation-induced cell death. The combination of irradiation and quercetin treatment aggravated DNA damages and caused typical apoptotic cell death; as well the expression of Bax and p21 elevated and the expression of Bcl-2 decreased. Knocking down of p53 could reverse all the above effects under quercetin in combination with radiation. In addition, quercetin-induced radiosensitization was through stimulation of ATM phosphorylation. In human ovarian cancer xenograft model, combined treatment of quercetin and radiation significantly restrained the growth of tumors, accompanied with the activation of p53, CCAAT/enhancer-binding protein homologous protein, and γ-H2AX. Overall, these results indicated that quercetin acted as a promising radiosensitizer through p53-dependent ER stress signals.

Keywords: ATM kinase; DNA double-strand breaks; eIF-2α (eukaryotic initiation factor 2α); endoplasmic reticulum stress; p53; quercetin.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Effect of quercetin on the expression levels of DSB repair related proteins. Notes: (A) OV2008 cells were treated with quercetin at the indicated concentrations for 12 h. After incubation, cell extracts were analyzed by Western blotting. Actin was used as a loading control. (B) OV2008 cells were treated with vehicle, quercetin (100 μM), tunicamycin (5 μM), or quercetin (100 μM) in the presence of the ER stress inhibitor 4-PBA (10 μM) for the indicated times. After incubation, cell extracts were analyzed by Western blotting. Actin was used as a loading control. (C) OV2008 cells were treated with quercetin (100 μM) in the presence or absence of 4-PBA (10 μM), or the ER stress inducer tunicamycin (5 μM), for 12 h as indicated. After incubation, cell extracts were analyzed by immunofluorescence microscopy conducted using anti-Rad51 staining. Shown are mean foci per nucleus counted from three independent experiments. *p<0.05, when compared with control. Abbreviations: 4-PBA, 4-phenyl butyrate; DAPI, dihydrochloride; DSB, double-strand break; ER, endoplasmic reticulum.
Figure 2
Figure 2
Effect of quercetin on cellular sensitivity to IR. Notes: Cells were pretreated with or without quercetin (100 μM) for 12 h, followed by X-irradiation. (A) The cellular sensitivity to IR was assessed by clonogenic survival assay. (B) Cells were either sham irradiated or exposed to 4 Gy IR 1 h after pretreatment with vehicle or 100 μM quercetin. DNA damage was seen in the form of a comet tail under a fluorescence microscope (left) and it was quantified (right). For each comet image, PI intensities of the head and tail portions were obtained, and the percentage intensity of the tail portion was multiplied by the size of the tail (DNA migration) to yield a tail moment. *p<0.05, when compared with control. (C) Cells were treated with 4 Gy of irradiation (in the presence or absence of quercetin) and harvested at 12 h. Immunofluorescence microscopy was conducted using anti γ-H2AX or Rad51 staining. Shown are mean foci per nucleus counted from three independent experiments. *p<0.05, when compared with control. Abbreviations: DAPI, dihydrochloride; IR, ionizing radiation; PI, propidium iodide; Qu, quercetin.
Figure 3
Figure 3
Quercetin increases levels of p53, phospho-p53 (serine 15, 20), and sensitizes ovarian cancer cells to DSB in response to irradiation. Notes: (A and B) Scrambled siRNA and p53 siRNA were transfected into the cells. Then, cells were pretreated with or without quercetin (100 μM) for 12 h, followed by X-irradiation. The cellular sensitivity to IR was assessed by clonogenic survival assay. (C and D) Cells were treated with 4 Gy of IR in the presence or the absence of quercetin (100 μM, 1 h before IR and maintained for 24 h). After incubation for 48 h, cell extracts were analyzed by Western blotting. Actin was used as a loading control. (E and F) Scrambled siRNA and p53 siRNA were transfected into the cells. After incubation, cell extracts were analyzed by Western blotting. Actin was used as a loading control. (F) Immunofluorescence microscopy was conducted using anti γ-H2AX (left) or Rad51 (right) staining. Shown are mean foci per nucleus counted from three independent experiments. Errors bars represent standard deviation. Abbreviations: Con, control; DAPI, dihydrochloride; DSB, double-strand break; IR, ionizing radiation; Qu, quercetin.
Figure 4
Figure 4
p53 plays a crucial role in enhancing the apoptotic effect of quercetin in ovarian cancer cells treated with IR. Notes: (A) Cells were treated with indicated concentrations of quercetin for 24 h. The protein expressions of CHOP and p53 were analyzed by Western blot. (B and C) Cells were treated with IR (4 Gy) combined with quercetin (100 μM, 1 h before IR) followed by the presence or absence of the p53 inhibitor pifithrin-α (20 μM) for 24 h; Flag-p53 was transfected into the cells treated with IR (4 Gy) combined with quercetin (100 μM, 1 h before IR) followed by the presence of the p53 inhibitor pifithrin-α (20 μM) for 24 h; p53 siRNA was transfected into the cells followed by IR (4 Gy) combined with quercetin (100 μM, 1 h before IR) and maintained for 24 h. (B) Total cell lysates were subjected to Western blot analysis with specific antibodies to p53, CHOP, p-eIF2α, and eIF2α. (C) Western blot analysis for the detection of expression of Bcl-2 and cleaved caspase-12. (D) Flow cytometric analysis. Cells treated as in (B) were stained for the determination of annexin V/PI-positive populations using flow cytometry. (E) Flow cytometric analysis. Cells treated IR (4Gy) combined with quercetin (100 μM, 1 h before IR) followed by in the presence or absence of 4-PBA for 24 h, which were stained for the determination of annexin V/PI positive populations using flow cytometry. *p<0.05, when compared with control. Abbreviations: CHOP, CCAAT/enhancer-binding protein homologous protein; Con, control; FITC, fluorescein isothiocyanate; IR, ionizing radiation; PI, propidium iodide; QU, quercetin.
Figure 5
Figure 5
Quercetin targets ATM specifically in DDRs. Notes: (A) Cells treated with mock or IR (4 Gy) in the presence or absence of quercetin (100 μM, 1 h before IR) were harvested 2 h after IR and subjected to Western blotting with indicated antibodies. (B) Cells treated with IR (4 Gy) combined with quercetin (100 μM, 1 h before IR) or with the ATM inhibitor KU-55933 (20 μM, 2 h before IR) or with the DNA-PKcs inhibitor NU7026 (20 μM, 2 h before IR) were harvested 2 h after IR and subjected to Western blotting with indicated antibodies. (C) The ATM-deficient fibroblast cell line GM9607 was treated with indicated doses of radiation in the presence or absence of quercetin (100 μM) followed by the colony formation assay. Abbreviations: DDR, DNA damage response; IR, ionizing radiation; QU, quercetin.
Figure 6
Figure 6
In vivo antitumor effects of quercetin treated with IR in OV2008 xenografts. Notes: (A and B) OV2008 tumor cells were implanted into athymic nude mice. Mice received (2 mg/day, once daily for 14 doses) quercetin by oral gavage with/without radiotherapy (quercetin was given 1 h before IR) at 2 Gy fractions for a total dose of 20 Gy. (A) Effect of IR, and quercetin combined with or without IR on ovarian cancer growth, measurements of tumor volumes (left) or weights (right); *p<0.05, when compared with control. (B) Expression of p53, p-eIF2α, and γ-H2AX in tumor tissues of the OV2008 xenograft mouse model. Abbreviations: IR, ionizing radiation; Qu, quercetin; W, week.
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